Heteronuclear NMR relaxation phenomena can potentially provide detailed information about the dynamic behavior of internuclear vectors with macromolecules such as proteins. Relaxation of 13C nuclei due to dipolar interactions with bonded hydrogen(s) is of particular value because of the potential to report on the dynamic behavior of C-H vectors of the main chain and of both buried and surface amino acid side chains of proteins. In principle, the interpretation of relaxation for aliphatic carbons is straightforward owing to the dominant contribution of dipolar interactions to the observed relaxation rates. Unfortunately, however, the application of 13C relaxation measurements to studies of proteins has been hindered by the apparent requirement to selectively and specifically label individual amino acid residues of the protein under study. We have developed a simple pulse sequence that allows for the use of randomly fractionally 13C-enriched proteins. This experiment offers access to relaxation parameters from methine and methyl carbon sites throughout the protein using a single, easily prepared sample without the need for elaborate chemical synthesis or highly developed bacterial expression systems. A major concern in using uniformly 13C-enriched proteins for relaxation studies of the dynamics of proteins is the contribution of both 13C-1H and 13C-13C dipolar interactions to the observed relaxation behavior. Such a case would lead to multiexponential behavior making analysis of the observed relaxation time courses difficult and unreliable. We have randomly and fractionally enriched calmodulin with 13C by expression of the protein in E. coli during growth on a minimal media utilizing a mixture of labeled and unlabeled acetates (20% 13C2-acetate, 20% 13C1-acetate, 60% 12C-acetate) as the sole carbon source. At this level of fractional enrichment, carbon sites with one bonded carbon will have a 20% chance of being bonded to a 13C nucleus and sites with two bonded carbons will have a 40% chance of being bonded to at least one 13C nucleus. To suppress all 13C-13C pairs, the pulse sequence incorporates a low pass filter based on 13C-13C J-coupling. This sequence is designed to polarize the 13C-nuclei via the NOE, utilizes a difference T1 time course and increases sensitivity by use of a reverse B00 ir INEPTH033sequence. In the present case, this sequence has several distinct advantages over the more recent double B00 sequences used to study methineH035carbon or amide nitrogen relaxation. Simple polarization with the NOE avoids creation of unwanted and complicating 13C-13C coherences and allows use of 1H decoupling throughout the low pass filter with similar advantages.